MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification

A major challenge in proteomics is the absolute accurate quantification of large numbers of proteins. QconCATs, artificial proteins that are concatenations of multiple standard peptides, are well established as an efficient means to generate standards for proteome quantification. Previously, QconCATs have been expressed in bacteria, but we now describe QconCAT expression in a robust, cell-free system. The new expression approach rescues QconCATs that previously were unable to be expressed in bacteria and can reduce the incidence of proteolytic damage to QconCATs. Moreover, it is possible to cosynthesize QconCATs in a highly-multiplexed translation reaction, coexpressing tens or hundreds of QconCATs simultaneously. By obviating bacterial culture and through the gain of high level multiplexing, it is now possible to generate tens of thousands of standard peptides in a matter of weeks, rendering absolute quantification of a complex proteome highly achievable in a reproducible, broadly deployable system. One of the major challenges in proteomics is absolute quantification of individual proteins. The predominant technology in large scale protein quantification is MS of (usually tryptic) peptides derived from proteolysis of the proteome in vitro and it is well understood that although mass spectrometers can deliver linearity of response over many orders of magnitude, the response factor (signal intensity per mol of peptide) varies considerably among individual peptides (1, 2). One outcome is that commonly used “label-free” methods that sum the precursor ion intensities for the peptides derived from a single protein, are excellent for relative quantification, but are less satisfactory for absolute quantification. MS-based absolute quantification of proteins can be supported by external standards that are analyzed before and/or after the analyte or by stable-isotope labeled internal standards that are coanalyzed and which define the response factor for each peptide (3). These peptides can be individually synthesized and quantified (4) and there have been some remarkable large-scale studies. However, large numbers of accurately quantified peptides are costly. Further, a commercially produced, accurately quantified standard peptide is a finite resource and is hence best focused on low numbers of assays of a small number of target proteins. Intact protein standards (5⇓–7), or large fragments (8) provide multiple potential peptides for quantification of the targets. In 2005, a novel approach to the creation of standard peptides by biosynthesis was proposed in the form of QconCATs (9⇓⇓⇓–13). QconCATs are artificial proteins that are concatenations of standard peptides from multiple natural proteins, sometimes interspersed by short peptides to recapitulate the primary sequence context of the natural counterpart (14, 15). Peptides suitable for quantification are referred to as Q-peptides, and are not synonymous with proteotypic peptides, as the latter term refers to peptides, unique to one protein, that drive protein identification, not quantification. QconCATs genes are synthesized de novo and are routinely expressed in E. coli cultured in media supplemented with appropriate stable isotope labeled amino acids, such that peptides derived from QconCATs are discriminable from natural peptides within the mass spectrometer. The purified QconCATs are mixed with the biological analyte sample and coproteolyzed to generate a mixture of labeled (standard) and unlabeled (analyte) peptide pairs that can be analyzed by liquid chromatography coupled to MS to yield absolute quantification of the analyte proteins. QconCATs have the added advantage that with appropriate control of proteolysis (11) all standards are, by definition, in a 1:1 ratio, rendering independent quantification of each standard unnecessary; a single common peptide can function to quantify the QconCAT (13). However, successful expression of novel QconCATs in E. coli is not always guaranteed. In a large-scale quantification project that used over 100 independently designed and expressed QconCATs, we discovered that ∼1 in 10 of the concatamers would fail to express, even when a range of expression conditions were explored. Further, at a low frequency, some QconCATs were prone to proteolysis in the bacterial cell or during purification, rendering them of reduced value for quantification. Effective QconCAT deployment across large scale proteome quantification studies would require a high level of confidence in expression of every new construct. In addition, living-cell based synthesis systems are not ideal for high-throughput preparation of multiple QconCATs and many mass spectrometry laboratories are not equipped for the basic molecular biology that would be needed to subclone and express recombinant proteins. To enhance the potential of QconCAT technology for large-scale proteome quantification, we here focus on a wheat germ cell-free protein synthesis system (WGCFS)1 as a major enhancement to the workflow of high throughput QconCAT synthesis. WGCFS, which uses the powerful translation system for germination stored in wheat germ, realizes the highest yield of translation among commercially available eukaryotic derived cell-free systems (16⇓⇓⇓–20). Using WGCFS, we previously demonstrated the feasibility of synthesis of single, small QconCATs, typically 25 kDa (21). In the present study, we first assessed whether WGCFS could be used to express more typical QconCATs at approx. 60 kDa (for quantification of ∼25 proteins at two peptides per target protein), whether WGCFS would rescue “failed” QconCATs and whether this cell free system was able to reduce the risk of proteolytic degradation. Further, we established whether an additional step in efficiency could be derived from coexpression of multiple QconCATs in a single WGCFS reaction

Synthetic biology meets proteomics: Construction of a la carte QconCATs for absolute protein quantification

We report a new approach to the assembly and construction of QconCATs, quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. The new approach is based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. It offers a major gain in flexibility of QconCAT implementation and enables rapid and efficient editability that permits, for example, substitution of one peptide for another. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short also allows a second cycle of assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. We refer to this approach as the ALACAT strategy as it permits a la carte design of quantification standards

PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics

Unraveling acetogen gas fermentation using quantitative systems biology

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Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3−/− mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3−/− mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3−/− mice. Lipid metabolism disorders in Sik3−/− mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice

A Potent Inhibitor of SIK2, 3, 3′, 7-Trihydroxy-4′-Methoxyflavon (4′-O-Methylfisetin), Promotes Melanogenesis in B16F10 Melanoma Cells

Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4′-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2) as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB)-specific coactivator 1 (TORC1). Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4′-O-metlylluteolin), efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in Ay/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2+/−; Ay/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2+/−; Ay/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4′-O-methylfisetin (4′MF) and found that 4′MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4′-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2+/− mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo

Quantitative assay of targeted proteome in tomato trichome glandular cells using a large-scale selected reaction monitoring strategy

Abstract Background Glandular trichomes found in vascular plants are called natural cell factories because they synthesize and store secondary metabolites in glandular cells. To systematically understand the metabolic processes in glandular cells, it is indispensable to analyze cellular proteome dynamics. The conventional proteomics methods based on mass spectrometry have enabled large-scale protein analysis, but require a large number of trichome samples for in-depth analysis and are not suitable for rapid and sensitive quantification of targeted proteins. Results Here, we present a high-throughput strategy for quantifying targeted proteins in specific trichome glandular cells, using selected reaction monitoring (SRM) assays. The SRM assay platform, targeting proteins in type VI trichome gland cells of tomato as a model system, demonstrated its effectiveness in quantifying multiple proteins from a limited amount of sample. The large-scale SRM assay uses a triple quadrupole mass spectrometer connected online to a nanoflow liquid chromatograph, which accurately measured the expression levels of 221 targeted proteins contained in the glandular cell sample recovered from 100 glandular trichomes within 120 min. Comparative quantitative proteomics using SRM assays of type VI trichome gland cells between different organs (leaves, green fruits, and calyx) revealed specific organ-enriched proteins. Conclusions We present a targeted proteomics approach using the established SRM assays which enables quantification of proteins of interest with minimum sampling effort. The remarkable success of the SRM assay and its simple experimental workflow will increase proteomics research in glandular trichomes